This invention relates to heparin conjugates and methods of making and using thereof. More particularly, this invention relates to heparin-bile acid conjugates, heparin-bile acid conjugates further including a targeting moiety, heparin-bile acid conjugates wherein the heparin is bonded to the bile acid through the 3-carbon of the bile acid, and heparin conjugates wherein heparin is covalently bonded to sulfonated moieties. Methods of using these conjugates for treating cancer are also described.
Heparin, due to its heterogeneous structure, can interact with many kinds of proteins. Heparin has anti-tumoral and anti-inflammatory activities as well as its well known anti-coagulant activity. R. Sasisekharan et al., Roles of heparan-sulphate glycosaminoglycans in cancer, 2 Nat. Rev. Cancer 521-528 (2002). Among the many sorts of proteins that interact with heparin are growth factors, which are key regulators for cell mitogenic activity. Growth factors usually bind with growth factor receptors and can modulate cell growth. In particular, vascular endothelial growth factor (VEGF) is a key protein in physiological angiogenesis (or neo-vascularization), or formation of new blood vessels. N. Ferrara et al., The biology of VEGF and its receptors, 9 Nat. Med. 669-676 (2003). Angiogenesis is a complex multi-step process involving endothelial cell activation, controlled proteolytic degradation of the extracellular matrix (ECM), proliferation and migration of endothelial cells, and formation of capillary vessel lumina. Diaz-Flores et al., 33 Anat. Histol. Embryol. 334-338 (2004).
Binding of growth factors to heparins or heparan sulfates is thought to have a crucial role in the modulation of activity of the high-affinity receptors. S. Colin et al., In Vivo Involvement of Heparan Sulfate Proteoglycan in the Bioavailability, Internalization, and Catabolism of Exogenous Basic Fibroblast Growth Factor, 55 Mol. Pharmacol. 74-82 (1999); I. J. Mason, The Ins and Outs of Fibroblast Growth Factors, 78 Cell 547-552 (1994); S. Tessler et al., Heparin Modulates the Interaction of VEGF165 with Soluble and Cell Associated flk-1 Receptors, 269 J. Biol. 12456-12461 (1994). Unfractionated heparin (UFH) or heparan sulfates promote basic fibroblast growth factor (bFGF) receptor dimerization and activation, thus enhancing cell growth. J. Schlessinger et al., Regulation of Growth Factor Activation by Proteoglycans: What Is the Role of the Low Affinity Receptors?, 83 Cell 357-360 (1995). Interestingly, it has been shown that low molecular weight heparin (LMWH; MW =4500-6000 Da), in contrast to UFH, can hinder binding of growth factors to their high-affinity receptors as a result of its smaller size. Indeed, in vitro heparin fragments of less than 18 saccharide residues reduce activity of VEGF, and fragments of less than 10 saccharide residues inhibit activity of bFGF. G. C. Jayson et al., Heparin Oligosaccharides: Inhibitors of the Biological Activity of bFGF on Caco-2 Cells, 75 Br. J. Cancer 9-16 (1997); S. Soker et al., Variations in the Size and Sulfation of Heparin Modulate the Effect of Heparin on the Binding of VEGF165 to its Receptors, 203 Biochem. Biophys. Res. Comm. 1339-1347 (1994).
Many anti-cancer drugs were designed as inhibitors of VEGF and its receptors. Bevacizumab (Avastin®) is an FDA-approved, anti-angiogenic drug that is representative of such VEGF inhibitors. Bevacizumab is a basic monoclonal antibody that binds the negatively charged receptor binding domain of VEGF and, therefore, can block the interaction between VEGF and VEGF receptors (Flk1, KDR). L. M. Ellis, Mechanisms of Action of Bevacizumab as a Component of Therapy for Metastatic Colorectal Cancer, 33 Semin. Oncol. S 107 (2006); E. Bergsland & M. N. Dickler, Maximizing the Potential of Bevacizumab in Cancer Treatment, 9 Oncologist 36-42 (2004).
LMWH can bind the heparin binding domain of VEGF. The sulfate groups of heparin can bind with positively charged amino acid residues, such as arginine, histidine, and lysine. A model of complexes formed between the heparin binding domain of VEGF and heparin or heparan sulfate predicts that sulfate and carboxylate groups of heparin contact these basic amino acid residues in the heparin-binding cleft of the VEGF protein. C. J. Robinson et al., VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase, 281 J. Biol. Chem. 1731-1740 )2006). However, there is no evidence that binding of heparin to VEGF results in an anti-angiogenic effect. Treatment of VEGF with either UFH or LMWH had no effect on tumor-associated angiogenesis in an experimental model of colon cancer metastasis in rat liver. S. M. Smorenburg et al., In Vivo Treatment of Rats with Unfractionated Heparin (UFH) or Low Molecular Weight Heparin (LMWH) Does Not Affect Experimentally Induced Colon Carcinoma Metastasis, 17 Clin. Exp. Metastasis 451-456 (1999).
VEGF comprises two main parts, a positively charged heparin binding domain (HBD; amino acid residues 111-165) and a negatively charged receptor binding domain (RBD; amino acid residues 8-109). B. A. Keyt et al., Identification of Vascular Endothelial Growth Factor Determinants for Binding KDR and FLT-1 Receptors. Generation of Receptor-selective VEGF Variants by Site-directed Mutagenesis, 271 J. Biol. Chem. 5638-56-46 (1996). Because HBD and RBD are in separate domains, even though heparin binds with HBD, the RBD maintains its structure.
It has been reported that HBD-deleted VEGF can bind to the VEGF receptor, but mitogenic activity is absent. B. A. Keyt et al., The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency, 271 J. Biol. Chem. 7788-7795 (1996). Thus, it was concluded that the HBD is critical to the cell growth activity of VEGF. RBD binding to VEGF cannot maintain cell growth.
In vitro experiments have suggested that the VEGF HBD-mediated interaction with neuropilin-1 (NP-1) increases the affinity of VEGF for KDR (VEGF receptor 2). Furthermore, the affinity of VEGF for the NP-1 extracellular domain is greatly enhanced by the addition of heparin. These results suggest that heparin mediates a successful interaction between VEGF and the receptor. H. Jia et al., Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling, 281 J. Biol. Chem. 13493-13502 (2006).
Finally, the VEGF receptor is a monomer before binding with the RBD of VEGF. When a VEGF ligand binds with the monomer receptor, then receptor dimerization can be initiated. After dimerization, two domains of the receptor experience conformational changes, resulting in a coiled structure. After all these steps, angiogenic signaling occurs. C. Ruch et al., Structure of a VEGF-VEGF receptor complex determined by electron microscopy, 14 Nat. Struct. Mol. Biol. 249-250 (2007).